Quest for a potent antimalarial drug lead: Synthesis and evaluation of 6,7-dimethoxyquinazoline-2,4-diamines

Article abstract of DOI:10.1016/j.bmc.2021.116018

Quinazolines have long been known to exert varied pharmacologic activities that make them suitable for use in treating hypertension, viral infections, tumors, and malaria. Since 2014, we have synthesized approximately 150 different 6,7-dimethoxyquinazoline-2,4-diamines and evaluated their antimalarial activity via structure-activity relationship studies. Here, we summarize the results and report the discovery of 6,7-dimethoxy-N⁴-(1-phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (20, SSJ-717), which exhibits high antimalarial activity as a promising antimalarial drug lead.

Full text of DOI:10.1016/j.bmc.2021.116018

Bioorg. Med. Chem. 33 (2021) 116018
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quest for a potent antimalarial drug lead: Synthesis and evaluation of
6,7-dimethoxyquinazoline-2,4-diamines
,
*
,
,d
Yuki Mizukawaa, Mayumi Ikegami-Kawaib 2, Masako Horiuchib, Marcel Kaiserc
,
Masayoshi Kojimaa, Seiki Sakanouea, Seiya Miyagie, Christian Nanga Chicke,
Hiroyuki Togashia, Masayoshi Tsubukib, Masataka Iharaa b, Toyonobu Usukie
,
,
,*,3
,*,1
Isamu Itoha
a Synstar Japan Co., Ltd., 2-9-46 Sakaecho, Odawara, Kanagawa 250-0011, Japan
b Faculty of Pharmaceutical Science, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan
c Medical Parasitology & Infection Biology, Swiss Tropical & Public Health Institute, Socinstrasse 57, 4000 Basel CH-4002, Switzerland
d University of Basel, Petersplatz 1, 4003 Basel CH-4003, Switzerland
e Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Quinazolines have long been known to exert varied pharmacologic activities that make them suitable for use in
treating hypertension, viral infections, tumors, and malaria. Since 2014, we have synthesized approximately 150
different 6,7-dimethoxyquinazoline-2,4-diamines and evaluated their antimalarial activity via structure-activity
relationship studies. Here, we summarize the results and report the discovery of 6,7-dimethoxy-N4-(1-phenyl-
ethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (20, SSJ-717), which exhibits high antimalarial activity as a
promising antimalarial drug lead.
Antimalarial drug
Quinazoline-2,4-diamines
SAR
Derivatization
1. Introduction
and ACT have emerged in southeast Asia between 2001 and 2018. To
combat this drug resistance, there is an acute need for new, highly
Alongside acquired immunodeficiency syndrome (i.e., AIDS) and
tuberculosis, malaria is one of the three major worldwide epidemic
diseases. Various species of malaria parasites have been identified,
including Plasmodium falciparum, P. vivax, P. malariae, and P. ovale.
These species are widely distributed in the tropical and subtropical re-
gions near the equator, including areas of sub-Saharan Africa, Asia, and
Latin America. A recent report by the World Health Organization indi-
cated that 228 million cases of malaria infection occurred worldwide in
2018, causing an estimated 400,000 deaths, 67% of which were children
under 5 years of age.1
effective drugs that are preferably both orally available and
inexpensive.1
Quinazolines have long been known to exert physiologic activities. A
several number of patent applications have been filed for the use of
quinazolines as antihypertensive2–7 and antiviral8,9 agents. In addition,
a report10 and patent11 have been published regarding their use as
antitumor agents. It was also reported that 6,7-dimethoxyquinazoline-
2,4-diamines (DMQDAs) exhibit highly selective antilysine methyl-
transferase activity.12–14 Another report indicated that quinazoline-2,4-
amines (QDAs) are effective in the treatment of leishmaniasis.15 Several
studies have examined the antimalarial activity of QDAs. For example,
Werbel and co-workers tested approximately 100 QDAs for activity
against P. falciparum.16 Nzila and co-workers reported that QDAs with
dihydrofolate reductase inhibitory activity also exhibited antimalarial
activity.17 Garcia-Bustos and colleagues assayed 2 million compounds
Chloroquine has been used for many years as an antimalarial drug,
but it is no longer effective due to the emergence of resistant
P. falciparum strains in many regions of the world. Current malaria
treatment relies largely on artemisinin and artemisinin-based combi-
nation drug therapy (ACT), although mutants resistant to artemisinin
* Corresponding authors.
E-mail addresses: m-kawai@hoshi.ac.jp (M. Ikegami-Kawai), t-usuki@sophia.ac.jp (T. Usuki), isamuito@peach.ocn.ne.jp (I. Itoh).
For I.I.: For general and chemistry: Synstar Japan Co., Ltd. Sakaecho, Odawara, Kanagawa 250-0011, Japan.
For M.I.-K.: For in vivo studies: Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan.
1
2
3
For T.U.: For general and chemistry: Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan.
https://doi.org/10.1016/j.bmc.2021.116018
Received 14 December 2020; Received in revised form 6 January 2021; Accepted 8 January 2021
Available online 16 January 2021
0968-0896/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Table 1
Effect
of
6,7-dimethoxy
groups
and
2-position
functional
group
on
antimalarial
activity
and
cytotoxicity
of
QDAs
.
Compound
IC50 (nM)a,c
IC50 (µM)b,cL6 rat myoblasts
Selectivity index (SI)d
P. falciparum NF54
1
2
3
4
5
6
2.10 ± 0.5
121 ± 16
3.67 ± 0.12
3.01 ± 0.57
5.29 ± 0.16
52.6 ± 4.8
67.3 ± 1.4
73.6 ± 5.5
1,750
25.6
230
23.0 ± 6.6
15,800 ± 2,600
34,800 ± 6,000
4,710 ± 230
6.25
3.33
1.90
15.6
Chloroquine (control)
podophyllotoxin(control)
0.0145
a
IC50 values for inhibition of P. falciparum NF54 growth. b IC50 values for inhibition of rat L6 cell growth. c Data are expressed as the average of two independent
determinations of IC50 ± deviation from the mean. d SI is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P. falciparum).
selected from a GSK library for in vitro activity against P. falciparum and
identified 8,000 compounds that exhibited 80% inhibition of
P. falciparum at a concentration of 2 µM, ultimately obtaining 44 anti-
malarial drug leads that included two DMQDAs.18 Fuchter and co-
workers reported that DMQDA with a six-membered ring amino group
(piperidine, piperazine) or a 7-membered ring amino group at the 2-po-
sition of the quinazoline ring exhibits selective antilysine methyl-
transferase activity and potent antimalarial activity in vitro.19,20 Finally,
Sleebs et al. reported the results of a detailed analysis of the antimalarial
activity of 56 types of QDAs containing various aniline derivatives at the
2-position of the quinazoline ring.21
indicate that the dimethoxy groups play an important role in the anti-
malarial activity of QDAs. Thus, we decided to prepare various QDAs
with 6,7-dimethoxy groups.
We next focused on the effect on antimalarial activity of an amino
group at the 2-position. Three compounds (3, 4, and 5) with an ethyl-
amino substituent at the 4-position of the 6,7-dimethoxyquinazoline
were synthesized. Although compound 3 with n-butylamino group at
the 2-position exhibited good activity, no activity was observed with a
chlorine atom (4), or a methoxy group (5) at the 2-position (Table 1).
Compound 6, which has no substitution at the 2-position, exhibited only
low activity. These results indicate that the presence of an amino group
at the 2-position of the 6,7-dimethoxy-quinazoline skeleton has a sig-
nificant effect on antimalarial activity.
Since 2014, we have been engaged in the development of orally
available antimalarial drugs, with particular attention centered on
DMQDAs. To date, we have synthesized approximately 150 compounds
and screened their in vitro antimalarial activity. Some promising com-
pounds demonstrating high potency have advanced to in vivo evalua-
tions using malaria-infected mice. Here, we report the chemical
synthesis and antimalarial activity of these DMQDAs in detail. Our ef-
forts have culminated in the discovery of a promising compound
exhibiting high antimalarial activity, 6,7-dimethoxy-N4-(1-phenyl-
ethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (20), which is expected to
become a promising antimalarial drug lead.
Based on these results, we synthesized ca. 150 DMQDAs with the
goal of identifying promising antimalarial drug candidates. Each syn-
thesis was carried out in two steps using commercially available 2,4-
dichloro-6,7-dimethoxyquinazoline as the common starting material
(Scheme 1). Because the chlorine atom at the 4-position is more reactive
than the chlorine atom at the 2-position, first substitution with the
chosen amine (R2) at 30–50 ◦C gave a 2-chloro-N4-(R2)-quinazolin-4-
amine intermediate. The solution containing the intermediate was
added to a solution containing the second amine (R1), and the reaction
temperature was increased to 130–150 ◦C to allow substitution of the R1-
amino group into the 2-position of the 6,7-dimethoxyquinazoline
skeleton.
2. Results and discussion
2.1. Synthesis of DMQDAs
2.2. In vitro antiplasmodial activity and cytotoxicity
In developing QDA-based antimalarial drugs, we first addressed the
necessity of the two methoxy groups at the 6- and 7-positions of the QDA
skeleton by investigating their effects on antimalarial activity and
cytotoxicity. In vitro antimalarial assays were conducted using
P. falciparum NF54, whereas rat-derived L6 cells were used for cyto-
toxicity assays. The selectivity index (SI) was determined by dividing the
IC50 value for cytotoxicity by the IC50 value for antimalarial activity.
Compound 1, which has methoxy groups at the 6- and 7-positions of
the N2,N4-di-n-butyl-quinazoline-2,4-diamine, exhibited 60-fold higher
antimalarial activity (against P. falciparum NF54) than compound 2,
which has no 6,7-dimethoxy groups (Table 1), whereas the cytotoxicity
(against L6 rat myoblasts) of the compounds was similar. The antima-
larial activity of 1 was also higher than that of chloroquine. These results
The antimalarial activity (against P. falciparum NF54) and cytotox-
icity (against L6 rat myoblasts) were also tested in vitro. The selectivity
index (SI) was calculated from the values thus obtained. Table 2 shows
the data for 40 compounds as representatives of the 150 synthesized
products. Twenty-nine compounds exhibited high antimalarial activity,
with IC50 values < 30 nM, which was stronger than values reported for
QDA.7–12 Compound 18, having 2- and 4-amino groups without an
active hydrogen, exhibited extremely low antimalarial activity (IC50
>
1,000 nM), which was also the case for compound 14, which has an NH2-
group at the 4-position. These results suggest that an amino group
containing one active hydrogen at the 4-position of the 6,7-dimethoxy-
qunazoline is essential for antimalarial activity. Sixteen compounds
2

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
exhibited potent activity (IC50 values < 10 nM),7–12 all of which were
substituted at the 2-position with an alkylamino group (1 and 7), (pyr-
idin-3-ylmethyl)amino group (12), (pyridin-4-ylmethyl)amino group
(13), pyrrolidin-1-yl group (15–19, 20, and 21), (pyridin-4-yl)amino
group (24–27, 29, and 30), or (pyridin-3-yl)amino group (35). In
contrast, compound 8, which has an alkoxyamino group at the 2-posi-
tion, exhibited less-potent activity. Compounds 9 and 10, which have
benzylamino and methylbenzylamino groups at the 2-position, exhibi-
ted IC50 values > 10 nM. In addition, compounds 22, 23, 31–34, and
36–39 exhibited antimalarial IC50 values > 10 nM. Thus, with respect to
inhibitory activity against P. falciparum NF54, there was no significant
difference between compounds with amino substituents at the 2-posi-
tion. However, eight compounds with a pyrrolidin-1-yl (16), (pyridin-
3-ylmethyl)amino (12), pyridin-4-yl (24–27, and 30), or (pyridin-3-yl)
amino (35) group exhibited better inhibitory profiles, with SI values >
3,000. With the exception of 16, these compounds all have a pyridine
moiety at the 2-position. Thus, installation of a pyridine ring at the 2-po-
sition is an effective means of significantly reducing cytotoxicity.
Although compounds having a pyrrolidin-1-yl group at the 2-posi-
tion demonstrated high antimalarial activity, most compounds did not
exhibit a particularly good SI value (i.e., <1000) because of their rela-
tively high cytotoxicity against L6 cells. Compounds having a different
substituent, such as an alkyl group (1, 3, and 7) or benzyl group (9), at
the 2-position exhibited potent to moderate antimalarial activity but
relatively high cytotoxicity against L6 cells. In contrast, compound 40,
which was reported by Garcia-Bustos and co-workers,18 exhibited lower
activity.
29–31, 33, and 34), compounds 29 and 31 exhibited an inhibition rate
of > 50% after 4 days. Contrary to our expectations based on the in vitro
assay results, however, even the best compound, 31, exhibited only a
60.8% inhibition rate. These results suggest that the presence of a pyr-
idine ring at the 2-position of the 6,7-dimethoxyquinazoline increases
the overall water solubility of the molecule, thus making it too hydro-
philic and consequently either impeding adequate translocation of the
molecule through the small intestine into the bloodstream or possibly
facilitating overly rapid metabolism in the blood stream. The former
assumption would also explain why most of the N2–(pyridin-4-yl)amino
compounds exhibited such low cytotoxicity in the in vitro assay (i.e.,
difficulty in entering L6 cells due to high hydrophilicity).
Figure 1 shows the correlation between the C log P of the six com-
pounds (15, 17, 20–23) having a pyrrolidin-1-yl group at the 2-position
and the mean malarial activity suppression rate of the corresponding
hydrochloride salts. An optimum C log P value of 4.5 was observed,
indicating that the in vivo activity of these compounds is affected by their
hydrophobicity/hydrophilicity balance. However, no correlation was
observed between inhibition rate and C log P for the group of pyridine-
containing compounds, as listed in Table 3.
As the preliminary results shown in Table 3 indicated that the 2-
(pyrrolidin-1-yl) compounds are highly active antimalarial agents, in
vivo studies were conducted using compounds 15, 20, and 23. Animals
were dosed with 50 mg/kg of the free form, 50 mg of free form per kg
equivalent of the hydrochloride salt, or 100 mg of free form per kg
equivalent of the hydrochloride salt, using five mice in each assay,
including the control. The parasitemia (%) and MSD values were
compared against the control group using the Student’s t-test unless
otherwise indicated. The results are summarized in Table 4. Compound
15 exhibited a 60% suppression rate at a dose of 50 mg/kg of free form
(vs control, p < 0.05), whereas its corresponding hydrochloride
exhibited a much better suppression rate of up to 96% (free form vs
control, p < 0.05) and a dramatic increase in MSD from 6.0 days (control
group) to 17.5 days. In addition, dosing of the hydrochloride salt at 100
mg/kg free-form equivalent resulted in potent antimalarial activity,
such that no infected blood cells were found on day 4 (vs control, p <
0.01); however, the MSD was shortened to 11.6 days, contrary to our
expectations. Although there was no significant difference between the
antimalarial activity of the free form and corresponding hydrochloride
salt at a dose of 50 mg/kg (free-form equivalent), compound 20 (SSJ-
717) exhibited potent antimalarial activity (90.4% and 92.1% suppres-
sion rate, respectively) and an MSD of approximately 20 days (vs con-
trol, p < 0.01). Mice dosed with 100 mg/kg free-form equivalent of 20
2.3. In vivo efficacy studies
For the in vivo assay, we used a rodent malaria parasite (P. berghei) as
an animal model that has a very similar lifecycle to that of P. falciparum.
Blood of mice infected with P. berghei NK65 was collected and used to
infect other mice (SLc: ICR females 25–27 g, n = 3 or 5) via tail vein
injection. The test compounds were suspended in 0.5% methylcellulose
aqueous solution and administered orally once per day for 4 days. In
order to enhance the bioavailability of the compounds, corresponding
hydrochloride salts of each test compound were prepared to increase the
water solubility. The number of HCl and H2O molecules in each hy-
drochloride salt was confirmed by elemental analysis, and the empirical
formula was determined. The oral dose was adjusted to a hydrochloride
salt equivalent of 50 mg of the free form per kg of mouse body weight.
Four days after infection, blood was collected from the tail of each
mouse and stained with Diff-Quik (Sysmex). Thin-layer smears were
prepared and observed under a microscope to calculate the suppression
of infection ratio (%) relative to that of the control group. The mean
survival days (MSD) of infected mice was compared to that of the control
group.
(SSJ-717) and the corresponding hydrochloride salt exhibited
a
remarkable suppression rate as high as > 99% (no infected blood cells
were observed) and an MSD of > 28 days (vs control, p < 0.01). It should
be noted that one of the five mice was completely cured, and this is the
first reported case of a malaria-infected mouse being cured by a quina-
zoline derivative. The effect of a salt dose equivalent to 50 mg/kg free
form on MSD was not examined for 23; however, the MSD was pro-
longed to 14.5 days with a salt dose equivalent of 100 mg/kg free form
(vs control, p < 0.05).
Based on the results of the in vitro studies described above, an in vivo
assay was conducted using 6,7-dimethoxy-2-(pyrrolidin-1-yl)quinazo-
lin-4-amines 15, 17, 20–23, and N2–(pyridin-4-yl)-2,4-diamino com-
pounds 24, 25, 29–31, 33, and 34 based on their high antimalarial
activity and very low cytotoxicity, which resulted in good SI values.
Table 3 shows the assay results obtained after dosing with hydrochloride
salts of the compounds equivalent to 50 mg of the free form per kg of
mouse body weight (n = 3; 3 mice were used in each case). The
empirical formula of each salt, including the number of attached HCl
and H2O molecules determined by elemental analysis, is also shown in
the table.
In addition, Kaplan-Meier survival analyses were carried out using
the MSD values of 15 and 20, and the results are shown in Figure 2.
Although 15 suppressed parasitemia by > 60% at a dose of 50 mg/kg, no
significant difference in MSD was observed versus the control. However,
the MSD value was markedly prolonged for the corresponding hydro-
chloride salt, as shown in Figure 2a (vs control, p < 0.05). No significant
differences in suppression rate or MSD were observed between free
compound 20 and the corresponding hydrochloride salt, but the MSD
value was prolonged in a dose-dependent manner to > 28 days (vs
control, p < 0.01, Figure 2b).
Six 2-(pyrrolidin-1-yl) compounds, 15, 17, and 20–23, which
exhibited fairly high antimalarial activity and some degree of cytotox-
icity in the in vitro assay, were selected. Four of the six compounds (15,
20, 22, and 23) exhibited inhibition rates > 80% (Table 3). In particular,
compounds 15 and 20 were highly active, with 15 exhibiting an inhi-
bition rate of 96.5% and 20 (SSJ-717) an inhibition rate of 99.6%. In
contrast, among the seven N2–(pyridin-4-yl)amino compounds (24, 25,
Our data suggest that these compounds 15 and 20 could be strong
inhibitors of histone lysine methyltransferase,6–7 resulting in potent
antimalarial activity.12 However, it has been reported that these com-
pounds might also inhibit human G9a.14 As such, the detailed
3

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Scheme 1. Synthesis of DMQDAs.
3.5. Purification method A:
mechanism underlying the activity of these compounds should be
investigated in the future. Development of a method to realize strong in
vivo activity for 2-(pyridin-4-yl) derivatives is another future subject for
research due to the potent in vitro activity of these compounds.
In summary, we synthesized approximately 150 DMQDAs and
evaluated their antimalarial activity. A total of 40 DMQDAs were sub-
sequently selected and examined in the present SAR study. Hydrochlo-
ride salts of DMQDAs having a pyrrolidin-1-yl group at the 2-position
(15, 20, and 23) exhibited improved transfer from the small intestine to
the blood when administered orally, resulting in strong in vivo antima-
larial activity. Compound 20 (SSJ-717) in particular was identified as a
potent lead compound capable of curing malaria in mice. Further SAR
studies and investigations of the mode of action of 20 are currently
underway in our laboratories.
The solid obtained according to general procedure B was heated and
stirred in EtOAc for 1–3 h at 90 ◦C and then filtered off after cooling to
room temperature to obtain the pure solid.
3.6. Purification method B:
The solid obtained according to general procedure B was recrystal-
lized in acetonitrile to afford the pure solid.
3.7. Purification method C:
In cases in which the purity of the 6,7-dimethoxyquinazoline-2,4-
diamine was < 96% after purification method A or B, the product was
purified again by column chromatography using silica gel (35–60 mesh)
and elution with a mixture of EtOAc and MeOH (90:10 to 60:40).
Nuclear magnetic resonance (NMR) spectra were recorded at room
temperature on a JEOL JNM-ECA600 II (600 MHz for 1H, 150 MHz for
13C) spectrometer or JEOL JNM-ECA 500 (500 MHz for 1H, 125 Hz for
13C) spectrometer in the solvent indicated. All 1H NMR chemical shifts
are reported in ppm (δ) downfield of TMS. All 13C NMR chemical shifts
are reported in ppm (δ) relative to the signals for chloroform (77 ppm),
DMSO (39.5 ppm), or MeOH (49.3 ppm) with 1H decoupled observation.
Data for 1H NMR are reported as follows: chemical shift (δ ppm), mul-
tiplicity (s = singlet, brs = broad singlet, d = doublet, dd = double
doublet, t = triplet, q = quartet, m = multiplet), integration, and J
coupling constant (Hz). Data for 13C NMR spectra are reported in
chemical shift (δ ppm). Mass spectrometry (MS) was performed on a
Xevo G2 TOF (Waters) in positive ion mode using an atmospheric solids
analysis probe. High-resolution MS (HRMS) spectra with electrospray
ionization (ESI) or fast atom bombardment (FAB) were obtained using a
JEOL JMS-T100LC or JEOL JMS-700 instrument and are reported in
terms of mass-to-charge ratio (m/z). HPLC analyses were performed on a
LC-10ADvp (Shimadzu) instrument with ultraviolet (UV) detector set to
254 nm. Samples were dissolved in MeOH or mixed solvent of MeOH
and ethylacetate (50:50) and injected onto a TSK-gel 100 V 5.0 µm, 4.6
mm × 150 mm column maintained at 40 ◦C. The flow rate was 1.0 mL/
min. A binary gradient of 0.1% acetic acid and 0.1% triethylamine in
MeOH (A) and H2O (B) at 0.01–3 min (hold: A/B = 50/50), 3–20 min
(linear gradient: A/B = 50/50 to A:100%), 20–25 min (A: 100%). All
final compounds were obtained at > 95% purity using the HPLC method
described above.
3. Experimental section
3.1. Chemistry
All reagents and solvents were obtained from FUJIFILM Wako Pure
Chemical or TCI and used without further purification. 2,4-Dichloro-6,7-
dimethoxyquinazoline and 2,4-dichloroquinazoline with 98% and 97%
purity, respectively, were purchased from FUJIFILM Wako Pure
Chemical.
3.2. General procedure a (for 2-chloro-6,7-dimethoxyquinazolin-4-
amines [intermediates]):
To a solution of commercially obtained 2,4-dichloro-6,7-dimethoxy-
quinazoline (1.04 g, 4 mmol), 2–2.5 equivalents of the selected amine in
acetonitrile (10 mL) was added, and the solution was heated and stirred
at 35–50 ◦C for 3–5 h. After insoluble solids were filtered off, the filtrate
was poured into water to afford the intermediates at 70–95% yield. The
purity of the obtained intermediates was > 95% (high-performance
liquid chromatography [HPLC] analysis), and they were used in the final
step without further purification.
3.3. General procedure B (for 6,7-dimethoxyquinazoline-2,4-diamines):
A mixture of the intermediates (2 mmol) and selected amine (5
mmol) in dimethylacetamide (DMA, 5 mL) was heated at 120–150 ◦C for
3–10 h, and the reaction mixture was then poured into water to afford a
crude solid. The solid was washed with water and dried at room tem-
perature and then purified using the methods described below to obtain
pure solids (purity > 96%). The yields after purification were 50–72%.
3.8. N2,N4-Di-n-butyl-6,7-dimethxyquinazoline-2,4-diamine (1):
3.4. General procedure C (for hydrochloride salt preparation):
To a solution of 2,4-dichloro-6,7-dimethoxyquinazoline (1.04 g, 4
mmol: Wako Chemicals) in acetonitrile (10 mL), n-butylamine (0.584 g,
8 mmol) was added at room temperature, and the mixture was stirred for
3 h at 35–40 ◦C. The reaction mixture was then poured into water (100
mL), and the precipitated solid was dried at room temperature to afford
0.931 g (79.0% yield) of N4-n-butyl-2-chloro-6,7-dimethoxyquinazolin-
4-amine.
To a solution of 6,7-dimethoxyquinazoline-2,4-diamines (2.5 mmol)
in MeOH (20 mL), 37% hydrochloride (HCl, 0.23 mL) was added
dropwise at 5–10 ◦C, and the mixture was stirred for 1 h at room tem-
perature. The reaction mixture was then concentrated under reduced
pressure. Ethylacetate (EtOAc, 150 mL) was added to the residue, and
the resulting mixture was stirred for 1 h at room temperature. The solids
were then filtered and washed with EtOAc to afford the HCl salts at
yields of 70–90%. The empirical formula of the salts, including attached
H2O molecules, was determined by elemental analysis (JM10, Yanaco
Technical Science).
A solution of N4-n-butyl-2-chloro-6,7-dimethoxyquinazolin-4-amine
(0.593 g, 2 mmol) and n-butylamine (0.365 g, 5 mmol) in DMA (5 mL)
was heated at 140–145 ◦C and stirred for 12 h. The reaction mixture was
then poured into water (100 mL), and the precipitated solid was
collected. After drying at room temperature, the solid was purified
4

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Table 2
In vitro antimalarial activity and cytotoxicity (IC50 values) of newly synthesized DMQDAs
.
Compound
R1
R2
IC50a,c (nM)P. falciparum
IC50b,c (µM) L6 cells
Selectivity index (SI)d
1
7
8
9
-NHnBu
-NHnBu
-NHiPr
-NHnBu
-NHEt
2.10 ± 0.5
3.14 ± 0.8
27.6 ± 5.2
14.8 ± 2.9
3.67 ± 0.12
5.31 ± 0.33
4.46 ± 0.20
5.44 ± 0.18
1,750
1,690
161
-NHnBu
–NH(CH2)3OEt
367
10
11
-NHEt
17.0 ± 0
1.68 ± 0.22
10.6 ± 0.77
99
-NHnBu
10.9 ± 1.3
1,000
12
13
-NHnBu
-NHnBu
2.17 ± 0
11.1 ± 0.63
11.0 ± 5.3
5,120
1,360
8.16 ± 4.0
14
15
16
17
18
19
–NH2
1,660 ± 84
1.80 ± 0
28.2 ± 1.5
61.8 ± 3.4
12.5 ± 0.63
1.48 ± 0.13
3.96 ± 0.93
9.49 ± 4.5
17.0
-NHnBu
-NHiPr
2,650
3,430
1,060
3.80
47.1 ± 11
1.40 ± 0.1
1,040 ± 15
5.49 ± 1.4
-NHnC8H17
1,900
20 (SSJ-717)
10.0 ± 1.3
4.81 ± 0.53
481
21
22
7.30 ± 3.7
13.6 ± 1.4
4.48 ± 0.39
27.3 ± 6.8
613
Me
-NHCH-CH2CH2
2,000
23
24
32.9 ± 4.1
3.07 ± 0
8.46 ± 2.3
55.1 ± 5.5
257
-NHEt
17,900
25
26
27
28
29
30
31
-NHiPr
2.90 ± 0.2
5.67 ± 2.8
2.83 ± 0
81.4 ± 13
105 ± 5.1
47.7 ± 5.8
110 ± 6.8
1.76 ± 0.63
62.7 ± 4.1
213 ± 45
28,000
5,300
16,800
2,130
241
-NHnBu
-NHCHMeEt
-NHCHEt2
-NHnC8H17
51.77 ± 2.7
7.30 ± 3.6
0.40 ± 0.10
49.0 ± 3.9
156,000
4,350
Me
-NHCH
(continued on next page)
5

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Table 2 (continued)
Compound
R1
R2
IC50a,c (nM)P. falciparum
22.6 ± 10
IC50b,c (µM) L6 cells
138 ± 11
Selectivity index (SI)d
6,100
32
33
12.8 ± 1.3
138 ± 18.4
10,700
34
35
30.9 ± 1.3
6.14 ± 1.5
138 ± 33
4,470
-NHEt
112 ± 0.77
18,200
36
37
38
39
-NHiPr
14.7 ± 1.5
25.4 ± 0
249 ± 35
150 ± 1.8
155 ± 27
170 ± 16
16,900
5,900
8,190
8,250
-NHnBu
-NHCHEt2
19.0 ± 1.4
20.6 ± 2.6
40
180 ± 60.6
4,980 ± 273
27.7
chloroquine (control for P. falciparum NF54)
6.25
podophyllotoxin (control for L6 cells)
0.0145
a
IC50 values for inhibition of P. falciparum NF54 growth.
b
c
IC50 values for inhibition of rat L6 cell growth.
Data are expressed as the average of two independent determinations of IC50 ± deviation from the mean.
SI is the ratio of IC50 for cytotoxicity versus antiplasmodial activity (L6/P. falciparum).
d
according to method A to obtain 0.545 g of 1 (82% yield); 1H NMR (600
MHz, CDCl3) δ 6.88 (s, 1H), 6.75 (s, 1H), 5.26 (s, 1H), 4.73 (s, 1H), 3.94
(s, 3H), 3.91 (s, 3H), 3.59 (t, J = 7.1 Hz, 2H), 3.47 (t, J = 7.1 Hz, 2H),
1.75–1.53 (m, 4H), 1.51–1.37 (m, 4H), 0.99–0.95 (m, 6H); 13C NMR
(150 MHz, CDCl3) δ 159.5, 159.3, 154.3, 148.9, 145.2, 105.7, 103.6,
100.8, 56.2, 55.9, 41.3, 40.9, 40.7, 32.2, 31.7, 20.3, 20.2, 13.91, 13.88;
ESI-MS (m/z) calcd for C18H29N4O2 [M + H]+ 333.23, found 333.29.
3.11. 2-Chloro-N4-ethyl-6,7-dimethoxyquinazolin-4-amine (4):
To a solution of 2,4-dichloro-6,7-dimethoxyquinazoline (1.04 g, 4
mmol) in acetonitrile (10 mL), ethylamine (0.43 g, 9.6 mmol) was added
at room temperature. The solution was heated and stirred at 35–40 ◦C
for 2 h. The reaction mixture was then poured into water (100 mL) to
afford a crude solid. The solid was purified according to method A to
obtain 0.88 g of 4 (82.2% yield); 1H NMR (600 MHz, DMSO‑d6) δ 8.34 (t,
J = 4.8 Hz, 1H), 7.61 (s, 1H), 7.06 (s, 1H), 3.91 (s, 6H), 3.51 (m, 2H),
1.24 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 159.8, 155.2,
154.3, 148.4, 147.1, 106.9, 106.5, 102.2, 56.1, 55.8, 35.6, 14.3; FAB-
HRMS (m/z) calcd for C12H15N3O2Cl [M + H]+ 268.0853, found
268.0854.
3.9. N2,N4-Di-n-butylquinazoline-2,4-diamine (2):
Starting with 2,4-dichloroquinazoline, the title compound was pre-
pared according to general procedures A and B and purified according to
method A (65% overall yield); 1H NMR (600 MHz, CDCl3) δ 7.65 (brs,
1H), 7.48 (q, J = 6.6, 1.2 Hz, 1H), 7.42 (brs, 1H), 7.06 (t, J = 6.6 Hz, 1H),
6.28–5.52 (brs, 2H), 3.59 (t, J = 7.2 Hz, 2H), 3.49 (t, J = 7.2 Hz, 2H),
1.67 (m, 2H), 1.59 (m, 2H), 1.42 (m, 4H), 0.96 (m, 6H); 13C NMR (150
MHz, CDCl3) δ 160.1, 158.9, 150.1, 132.7, 124.0, 121.2, 121.1, 110.8,
41.1, 40.9, 32.0, 31.3, 20.2, 20.1, 13.8, 13.8; ESI-MS (m/z) calcd for
C16H25N4 [M + H]+ 273.21, found 273.27.
3.12. N4-Ethyl-2,6,7-trimethoxyquinazolin-4-amine (5):
To a solution of 4 (0.534 g, 2 mmol) in DMA (10 mL), sodium
methoxide in MeOH (4 mmol) was added dropwise at 5–10 ◦C and
stirred for 4 h at room temperature. The reaction solution was then
poured into water to afford a crude solid. The solid was purified ac-
cording to method A to obtain 0.421 g of 5 (80% yield); 1H NMR (600
MHz, CDCl3) δ 7.09 (s, 1H), 7.04 (s, 1H) 4.03 (s, 3H), 3.99 (s, 6H), 3.71
(q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ 162.4, 160.6, 154.4, 148.0, 146.8, 106.6, 105.2, 100.5, 56.1, 56.1,
54.0, 36.2, 14.7; ESI-MS (m/z) calcd for C13H18N3O3 [M + H]+ 264.13,
found 264.19.
3.10. N2-n-Butyl-6,7-dimethoxy-N4-ethylquinazoline-2,4-diamine (3):
The title compound was prepared according to general procedures A
and B and purified according to method A (62% overall yield); 1H NMR
(600 MHz, CDCl3) δ 6.92 (s, 1H), 6.85 (s, 1H), 5.82 (brs, 1H), 5.20
(brs,1H), 3.93 (s, 3H), 3.90 (s, 3H), 3.63 (m, 2H), 3.47 (m, 2H), 1.60 (m,
2H), 1.43 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H), 0.94 (t, J = 7.2 Hz, 3H); 13
C
3.13. N4-n-Butyl-6,7-dimethoxyquinazolin-4-amine (6):
NMR (150 MHz, CDCl3) δ 159.2, 159.1, 158.5, 154.5, 145.4, 104.6,
103.4, 101.3, 56.3, 56.0, 41.2, 41.1, 36.1, 35.9, 32.1, 20.2, 14.8, 13.9;
ESI-MS (m/z) calcd for C16H25N4O2 [M + H]+ 305.20, found 305.26.
To a solution of commercially available 4-chloro-6,7-dimethoxyqui-
nazoline (674 mg, 3 mmol: TCI) in acetonitrile (5 mL), n-butylamine
6

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Table 3
Table 4
SAR of 6,7-dimethoxy-2-(pyrrolidin-1-yl)quinazolin-4-amines and 6,7-dime-
In vivo antimalarial activity of quinazoline derivatives and hydrochloride salts
thoxy-N2-(pyridin-4-yl)quinazolin-2,4-diamines in in vivo oral assays (n = 3).
Compound
administered via the oral route.
No.
C log
Pa
Empirical
formula of the
salt
Doseb
(mg/kg
× 4)
Suppressionc
(%)
MSDd
(days)
Form
Dose (mg/
Parasitemia
(%)
Suppression
(%)c
MSD
kg × 4)
(days)a
Control
0.5% MC
28.4 ± 10.2b
0.0
6.2 ±
Control
–
–
–
0.0
6.2
0.5b
2-(pyrrolidin-1-yl) compounds
15
3.83
5.50
4.64
5.34
2.99
5.05
C
18H26N4O2
HCl⋅H2O
22H34N4O2
HCl⋅H2O
22H26N4O2
HCl⋅H2O
24H30N4O2 1/
58.2
57.0
57.2
63.7
59.0
57.5
96.5
48.8
99.6
74.4
80.3
83.0
17.5
6.7
Free
H2O,
HCl
50
10.8 ± 4.9
1.5 ± 1.3
60.0
95.5
6.6 ± 1.1
17.5 ±
2.2
15
20
58.2 (50)
17
C
H2O,
HCl
116.4
(100)
NDd
>99
11.6 ±
1.4
20 (SSJ-
717)
C
12.0
11.7
8.0
21
C
Free
50
2.5 ± 1.3
2.0 ± 1.1
NDd
90.4
92.1
>99
19.8 ±
1.0
2HCl
22
23
C
20H23N5O2
H2O,
HCl
57.2 (50)
18.6 ±
2.2
HCl⋅3/2H2O
C21H24N4O2
14.7
H2O,
HCl
114.4
(100)
28.2 ±
HCl⋅H2O
1.7e
2-(pyridin-4-yl)amino compounds
24
25
29
30
2.46
2.77
5.03
4.17
C
17H19N5O2
58.4
58.2
58.4
63.7
1.0
7.0
8.3
7.3
8.3
Free
H2O,
HCl
50
23.4 ± 9.4
26.8
53.9
6.8 ± 1.2
7.0 ± 0.9
HCl⋅H2O
23
57.5 (50)
14.7 ± 11.1
C18H21N5O2
2HCl⋅5/2H2O
39.5
54.8
6.6
H2O,
HCl
115.0
(100)
11.6 ± 2.1
63.8
14.5 ±
C
23H31N5O2
4.5
HCl⋅2H2O
23H23N5O2
2HCl⋅2H2O
a
Mean survival days.
C
b
c
Average value of four experiments (19 mice).
Suppression on day 4 calculated from the reduction in parasitemia compared
31
33
4.57
2.51
C
22H21N5O2 HCl
54.7
66.3
60.8
27.1
7.3
8.7
C21H20N5O2
with each control group (n = 5 or 4).
d
2HCl⋅3H2O
No detection of infected blood cells.
e
34
2.51
C
21H20N5O2
66.3
29.4
7.7
Results included one censored case.
2HCl⋅3H2O
a
b
c
3.14. N2-n-Butyl-6,7-dimethoxy-N4-isopropylquinazoline-2,4-diamine
Calculated using ChemDraw.
Vehicle, 0.5% MC.
(7):
Mean suppression of parasitemia on day 4 (%) versus that of the control (n =
5).
The title compound was prepared according to general procedures A
and B, and purified according to method A (65% overall yield); 1H NMR
(600 MHz, CDCl3) δ 6.87 (s, 1H), 6.76 (s, 1H), 5.08 (brs, 1H), 4.73 (brs,
1H), 4.48 (m, 1H), 3.93 (s, 6H), 3.47 (m, 2H), 1.61 (m, 2H), 1.44 (m,
2H), 1.32 (s, 6H), 0.95 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
159.5, 158.5, 154.3, 149.0, 145.1, 105.8, 103.6, 100.9, 56.3, 55.9, 42.5,
41.2, 41.1, 32.2, 22.9, 22.8, 20.2, 13.9; ESI-MS (m/z) calcd for
d
Mean survival days (n = 3).
C
17H27N4O2 [M + H]+ 319.21, found 319.28.
3.15. N4-n-Butyl-6,7-dimethoxy-N2-(2-ethoxypropyl)quinazoline-2,4-
diamine (8):
The title compound was prepared according to general procedures A
and B, and purified according to method A (61% overall yield); 1H NMR
(500 MHz, CDCl3) δ 6.86 (s, 1H), 6.76 (s, 1H), 5.36 (brs, 1H), 5.01 (brs,
1H), 3.92–3.88 (m, 6H), 3.60–3.53 (m, 6H), 3.45–3.60 (m, 2H), 3.21 (s,
1H), 1.93–1.87 (m, 2H), 1.69–1.63 (m, 2H), 1.46–1.39 (m, 2H),
1.22–1.19 (m, 3H), 0.98–0.95 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 159.5, 159.4, 154.4, 149.0, 145.3, 105.8, 103.8, 101.0, 77.4,
77.2, 76.9, 69.0, 66.4, 56.3, 56.1, 41.0, 39.4, 37.2, 31.8, 30.2, 20.4,
15.4, 14.0; ESI-HRMS (m/z) calcd for C19H31N4O3 [M + H]+ 363.2396,
found 363.2396.
Figure 1. Correlation between C log P of 6,7-dimethoxy-2-(pyrrolidin-1-yl)-
quinazolin-4-amines and antimalarial activity of the corresponding hydro-
chloride salts.
(0.99 mL, 10 mmol) was added at room temperature, and the mixture
was heated for 5 h at 70–75 ◦C. Then, the reaction mixture was poured
into water (50 mL) and the precipitated solid was dried at room tem-
perature to afford 0.650 g of 6 (83% yield); 1H NMR (500 MHz, CDCl3) δ
8.44 (s, 1H), 7.06 (s, 1H), 6.82 (s, 1H), 5.59 (brs, 1H), 3.86–3.83 (m,
6H), 3.55–3.51 (m, 2H), 1.95 (s, 2H), 1.63–1.57 (m, 2H), 1.37–1.33 (m,
2H), 0.87–0.84 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 158.5,
154.3, 154.2, 148.99, 146.5, 108.7, 107.8, 99.6, 77.4, 77.2, 76.9, 56.3,
56.3, 41.4, 31.8, 20.4, 14.0; ESI-HRMS (m/z) calcd for C14H20N3O2 [M
+ H]+ 262.1556, found 262.1559.
3.16. N2-Benzyl-6,7-dimethoxy-N4-ethylquinazoline-2,4-diamine (9):
The title compound was prepared according to general procedures A
and B, and purified according to method A (58% overall yield); 1H NMR
(600 MHz, CDCl3) δ 7.39 (d, J = 6.6 Hz, 2H), 7.31 (m, 2H), 7.26 (m, 1H),
6.90 (s, 1H), 6.78 (s, 1H), 5.27 (s, 1H), 5.10 (s, 1H), 4.70 (d, J = 5.4 Hz,
2H), 3.94 (s, 3H), 3.91 (s, 3H), 3.59 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H); 13
C
NMR (150 MHz, CDCl3) δ 159.2, 159.1, 154.3, 148.6, 145.4, 140.2,
128.4, 127.5, 126.9, 56.2, 56.0, 45.6, 36.0, 14.8; ESI-MS (m/z) calcd for
C
19H23N4O2 [M + H]+ 339.18, found 339.24.
7

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Figure 2. Kaplan-Meier survival estimates for the antimalarial activity of compounds 15 and 20; (a) effect of the hydrochloride salt; (b) dose dependence.
3.17. 6,7-Dimethoxy-N4-ethyl-N2-(2-phenylethyl)quinazoline-2,4-
and B, and purified according to method C (58% overall yield); 1H NMR
(500 MHz, CDCl3) δ 8.63 (s, 1H), 8.47–8.46 (m, 1H), 7.71–7.69 (m, 1H),
7.22–7.21 (m, 1H), 6.85 (s, 1H), 6.80 (s, 1H), 5.54 (brs, 1H), 5.24 (brs,
1H), 4.70–4.68 (brs, 2H), 3.91 (s, 3H), 3.86 (s, 3H), 3.52–3.48 (m, 2H),
diamine (10):
The title compound was prepared according to general procedures A
and B, and purified according to method A (60% overall yield); 1H NMR
(500 MHz, CDCl3) δ 7.22–7.19 (m, 3H), 7.14–7.10 (m, 3H), 6.69 (s, 1H),
3.84–3.83 (m, 6H), 3.58–3.53 (m, 4H), 2.86–2.83 (t, J = 7.2 Hz, 2H),
2.05 (s, 3H) 1.29–1.26 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
179.7, 159.5, 155.3, 146.5, 139.2, 128.8, 128.6, 126.4, 103.1, 102.2,
77.4, 77.2, 76.9, 56.5, 56.4, 42.8, 36.8, 36.1, 24.7, 14.5; ESI-HRMS (m/
z) calcd for C20H25N4O2 [M + H]+ 353.1998, found 353.1990.
1.62–1.56 (m, 2H), 1.40–1.33 (m, 2H), 0.92–0.89 (t, J = 7.2 Hz, 3H); 13
C
NMR (125 MHz, CDCl3) δ 159.8, 159.3, 154.8, 149.6, 149.0, 148.6,
145.9, 136.4, 135.5, 123.7, 106.1, 104.3, 101.2, 77.7, 77.4, 77.2, 56.6,
56.4, 43.5, 41.3, 32.0, 20.6, 14.3; ESI-HRMS (m/z) calcd for C20H26N5O2
[M + H]+ 368.2087, found 368.2078.
3.20. N4-n-Butyl-6,7-dimethoxy-N2-(pyridin-4-ylmethyl)quinazoline-
2,4-diamine (13):
3.18. N4-n-Butyl-6,7-dimethoxy-N2-(pyridin-2-ylmethyl)quinazoline-
2,4-diamine (11):
The title compound was prepared according to general procedures A
and B, and purified according to method C (56% overall yield); 1H NMR
(500 MHz, DMSO‑d6) δ 8.43–8.42 (br s, 2H), 7.57 (br s, 1H), 7.38 (br s,
1H), 7.29–7.28 (d, J = 5.6 Hz, 2H), 6.88 (br s, 1H), 6.64 (s, 1H),
4.51–4.50 (d, J = 6.3 Hz, 2H), 3.78–3.77 (d, J = 5.7 Hz, 6H), 1.50 (br s,
2H), 1.29 (br s, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz,
DMSO‑d6) δ 159.2, 158.7, 153.7, 150.9, 149.2, 148.2, 144.6, 122.1,
103.2, 55.9, 55.3, 43.3, 40.0, 39.9, 39.7, 39.5, 39.4, 39.2, 39.0, 31.1,
19.9, 13.9; ESI-HRMS (m/z) calcd for C20H26N5O2 [M + H]+ 368.2087,
found 368.2093.
The title compound was prepared according to general procedures A
and B, and purified according to method A (56% overall yield); 1H NMR
(500 MHz, CDCl3) δ 8.54 (s, 1H), 7.61–7.57 (m, 1H), 7.37–7.35 (m, 1H),
7.14–7.12 (m, 1H), 6.88 (s, 1H), 6.79 (s, 1H), 5.72 (brs, 1H), 5.47 (brs,
1H), 4.83–4.82 (s, 2H), 3.92 (s, 3H), 3.87 (s, 3H), 3.54–3.47 (m, 2H),
1.61–1.55 (m, 2H), 1.40–1.34 (m, 2H), 0.92–0.89 (t, J = 7.2 Hz, 3H); 13
C
NMR (125 MHz, CDCl3) δ 159.9, 159.7, 159.5, 154.7, 149.3, 149.1,
145.7, 136.8, 122.1, 121.8, 106.1, 104.2, 101.2, 77.7, 77.4, 77.2, 56.6,
56.4, 47.4, 41.3, 32.0, 20.6, 14.3; ESI-HRMS (m/z) calcd for C20H26N5O2
[M + H]+ 368.2087, found 368.2111.
3.21. 6,7-Dimethoxy-2-(pyrrolidin-1-yl)quinazolin-4-amine (14):
3.19. N4-n-Butyl-6,7-dimethoxy-N2-(pyridin-3-ylmethyl)quinazoline-
The title compound was prepared according to general procedures A
and B, and purified according to method B (62% overall yield); 1H NMR
(600 MHz, CDCl3) δ 6.95 (s, 1H), 6.83 (s, 1H), 3.93 (s, 3H), 3.89 (s, 3H),
3.62 (m, 4H), 2.09 (s, 2H), 1.95 (m, 4H); 13C NMR (150 MHz, CDCl3) δ
2,4-diamine (12):
The title compound was prepared according to general procedures A
8

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
160.5, 157.8, 154.9, 150.2, 145.0, 105.4, 102.4, 101.4, 56.0, 46.6, 25.5;
365.1978, found 365.1994.
ESI-MS (m/z) calcd for C14H19N4O2 [M + H]+ 275.15, found 275.21.
3.27. 6,7-Dimethoxy-N4-(1-phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-
3.22. N4-n-Butyl-6,7-dimethoxy-2-(pyrrolidin-1-yl)quinazolin-4-amine
4-amine (20):
(15):
The title compound was prepared according to general procedures A
and B, and purified according to method B (68% overall yield); 1H NMR
(600 MHz, CDCl3) δ 7.43 (t, J = 8.4 Hz, 2H), 7.33 (t, J = 7.8 Hz, 2H),
7.26 (brs, 1H), 6.96 (s, 1H), 6.81 (s, 1H), 5.53 (m, 1H), 3.93 (s, 6H),
3.65–3.50 (m, 4H), 1.92 (m, 4H), 1.65 (d, J = 6.6 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 157.8, 154.3, 149.6, 144.8, 144.6, 128.4, 127.0,
126.3, 105.7, 102.7, 100.8, 56.3, 56.0, 50.2, 46.5, 25.5, 22.1; ESI-MS
(m/z) calcd for C22H27N4O2 [M + H]+ 379.21, found 379.29.
The title compound was prepared according to general procedures A
and B, and purified according to method B (57% overall yield); 1H NMR
(600 MHz, CDCl3) δ 6.93 (s, 1H), 6.75 (s, 1H), 5.23 (brs, 1H), 3.93 (s,
3H), 3.90 (s, 3H), 3.66–3.61 (m, 6H), 1.97–1.95 (m, 4H), 1.69 (m, 2H),
1.45 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
158.8, 157.9, 154.2 149.4, 144.7, 105.8, 102.8, 100.9, 56.2, 56.0, 46.5,
40.8, 40.7, 31.7, 25.6, 20.3, 13.9; ESI-MS (m/z) calcd for C18H27N4O2
[M + H]+ 331.21, found 331.28.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 61.08; H, 6.69; N, 12.88; Empirical
formula C22H26N4O2∙HCl⋅H2O: C, 61.03; H, 6.75; N, 12.94.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 56.12; H, 7.50; N, 14.56; Empirical
formula C18H26N4O2∙HCl⋅H2O: C, 56.17; H, 7.59; N, 14.56.
3.23. 6,7-Dimethoxy-N4-isopropyl-2-(pyrrolidin-1-yl)quinazolin-4-
3.28. 6,7-Dimethoxy-N4-(1-methyl-3-phenylpropyl)-2-(pyrrolidin-1-yl)
amine (16):
quinazolin-4-amine (21):
The title compound was prepared according to general procedures A
and B, and purified according to method B (68% overall yield); 1H NMR
(500 MHz, CD3OD) δ 7.28 (s, 1H), 6.90 (s, 1H), 4.91 (s, 4H), 3.89–3.86
(m, 6H), 3.60–3.51 (m, 6H), 1.98–1.95 (m, 4H), 1.74–1.72 (m, 2H),
1.00–0.97 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, CD3OD) δ 160.7,
159.2, 155.5, 149.5, 146.3, 105.2, 104.7, 104.0, 56.8, 56.1, 49.5, 49.3,
49.2, 48.8, 48.7, 48.5, 47.6, 43.8, 26.5, 23.6, 12.0; FAB-HRMS (m/z)
calcd for C17H25N4O2 [M + H]+ 317.1978, found 317.1959
The title compound was prepared according to general procedures A
and B, and purified according to method B (68% overall yield); 1H NMR
(600 MHz, CDCl3) δ 7.25 (d, J = 5.4 Hz, 3H), 7.18 (d, J = 7.8 Hz, 3H),
6.98 (s, 1H), 6.67 (s, 1H), 4.52 (t, J = 6.6 Hz, 1H), 3.96 (s, 3H), 3.92 (s,
3H), 3.61 (brs, 4H), 2.74 (m, 2H), 2.03 (m, 2H), 1.95 (m, 4H), 1.34 (d, J
= 6.6 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 158.1, 154.2, 147.8, 144.8,
142.0, 128.4, 128.4, 125.8, 105.7, 102.7, 100.9, 56.3, 56.0, 46.6, 46.3,
38.5, 32.7, 25.6, 20.8; ESI-MS (m/z) calcd for C24H31N4O2 [M + H]+
407.24, found 407.32.
3.24. 6,7-Dimethoxy-N4-octyl-2-(pyrrolidin-1-yl)quinazolin-4-amine
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 68.17; H, 7.12; N, 13.20; Empirical
formula C24H30N4O2∙1/2HCl: C,67.86; H,7.25; N,13.19.
(17):
The title compound was prepared according to general procedures A
and B, and purified according to method B (70% overall yield); 1H NMR
(600 MHz, CDCl3) δ 6.96 (s, 1H), 6.81 (s, 1H), 5.41 (brs, 1H), 3.93 (s,
6H), 3.66 (t, J = 6.6 Hz, 4H), 3.59 (m, 2H), 1.96 (m, 4H), 1.70 (m, 2H),
1.44–1.23 (m, 10H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ 158.8, 157.7, 154.1, 149.2, 144.8, 105.6, 102.8, 100.9, 56.2, 56.0,
46.5, 41.2, 31.8, 29.4, 27.2, 25.6, 22.6, 14.1; ESI-MS (m/z) calcd for
3.29. 6,7-Dimethoxy-N4-(pyridin-3-ylmethyl)-2-(pyrrolidin-1-yl)
quinazolin-4-amine (22):
The title compound was prepared according to general procedures A
and B, and purified according to method B (69% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 8.63 (s, 1H), 8.44 (d, J = 3.6 Hz, 1H), 8.32 (m,
1H), 7.79 (s, 1H), 7.45 (s, 1H), 7.35 (d, J = 4.8 Hz, 1H), 6.75 (s, 1H),
4.70 (d, J = 5.4 Hz, 2H), 3.85 (s, 3H), 3.80 (s, 3H), 3.45 (d, J = 6.6 Hz,
4H), 1.86 (t, J = 6.6 Hz, 4H); 13C NMR (150 MHz, CDCl3) δ 158.5, 157.1,
153.9, 149.2, 148.9, 147.9, 144.4, 136.0, 135.3, 123.4, 105.1, 102.9,
102.8, 55.8, 55.4, 46.1, 41.4, 25.1; ESI-MS (m/z) calcd for C24H31N4O2
[M + H]+ 407.24, found 407.32. ESI-MS (m/z) calcd for C20H24N5O2 [M
+ H]+ 366.19, found 366.27.
C
22H35N4O2 [M + H]+ 387.28, found 387.35.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 60.21; H, 8.40; N, 12.75; Empirical
formula C22H34N4O2∙HCl⋅H2O: C, 59.90; H, 8.46; N, 12.70.
3.25. 2,4-Bis(pyrrolidin-1-yl)-6,7-dimethoxyquinazoline (18):
The title compound was prepared according to general procedures A
and B, and purified according to method B (77% overall yield); 1H NMR
(500 MHz, DMSO‑d6) δ 8.44 (s, 1H), 7.06 (s, 1H), 3.82–3.77 (m, 10H),
3.49–3.46 (br t, 4H), 3.86–3.83 (m, 6H), 1.93–1.86 (m, 8H); 13C NMR
(125 MHz, DMSO‑d6) δ 159.3, 156.7, 153.4, 151.0, 143.0, 106.1, 105.2,
104.1, 55.6, 55.3, 49.9, 46.1, 25.3, 25.2; ESI-HRMS (m/z) calcd for
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 56.00; H, 6.34; N, 16.33; Empirical
formula C20H23N5O2∙HCl∙3/2⋅H2O: C, 55.68; H, 5.96; N, 16.23.
3.30. 6,7-Dimethoxy-N4-(2-methylphenyl)-2-(pyrrolidin-1-yl)
C
18H25N4O2 [M + H]+ 329.1978, found 329.1969.
quinazolin-4-amine (23):
3.26. N4-Benzyl-6,7-dimethoxy-2-(pyrrolidin-1-yl)quinazolin-4-amine
The title compound was prepared according to general procedures A
and B, and purified according to method B (70% overall yield); 1H NMR
(600 MHz, CDCl3) δ 8.07 (s, 1H), 7.25 (t, J = 7.2 Hz, 3H), 7.09 (d, J =
6.6 Hz, 1H), 7.00 (br, 1H), 6.89 (s, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 3.60
(s, 4H), 2.38 (s, 3H), 1.95 (t, J = 7.7 Hz, 4H); 13C NMR (150 MHz, CDCl3)
δ 157.2, 156.8, 154.7, 150.5, 145.2, 137.5, 130.4, 129.5, 126.4, 124.1,
123.3, 105.9, 103.0, 100.7, 56.2, 56.1, 46.6, 25.5, 18.2; ESI-MS (m/z)
calcd for C21H25N4O2 [M + H]+ 365.20, found 365.27.
(19):
The title compound was prepared according to general procedures A
and B, and purified according to method B (60% overall yield); 1H NMR
(500 MHz, CDCl3) δ 7.44 (d, J = 6.9 Hz, 2H), 7.36 (t, J = 7.4 Hz, 2H),
7.30 (s, 1H), 6.95 (s, 1H), 6.71 (s, 1H), 4.84 (d, J = 5.7 Hz, 2H), 3.96 (s,
3H), 3.89 (s, 3H), 3.65 (d, J = 6.3 Hz, 4H), 1.95 (d, J = 6.9 Hz, 4H); 13
C
NMR (125 MHz, CDCl3) δ 158.8, 158.0, 154.4, 149.8, 145.0, 139.7,
128.7, 128.2, 127.4, 105.9, 102.9, 101.0, 77.4, 77.2, 76.9, 56.3, 56.1,
46.7, 45.1, 25.7; ESI-HRMS (m/z) calcd for C21H25N4O2 [M + H]+
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 60.00; H, 6.42; N, 13.38; Empirical
formula C21H24N4O2∙HCl⋅H2O: C, 60.21; H, 6.50; N, 13.37.
9

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Bioorganic & Medicinal Chemistry 33 (2021) 116018
3.31. 6,7-Dimethoxy-N4-ethyl-N2-(pyridin-4-yl)quinazoline-2,4-diamine
3.36. 6,7-Dimethoxy-N4-octylamino-N2–(pyridin-4-yl)quinazoline-2,4-
diamine (29):
(24):
The title compound was prepared according to general procedures A
and B, and purified according to method C (64% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.03 (s, 2H), 7.79 (s, 1H), 7.08 (s, 1H), 6.84 (s,
The title compound was prepared according to general procedures A
and B, and purified according to method C (58% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.24 (d, J = 7.2 Hz, 2H), 8.99 (brs, 1H), 8.91 (d,
J = 4.8 Hz, 2H), 7.88 (s, 1H), 7.05 (d, J = 4.2 Hz, 2H), 3.92 (s, 6H), 3.62
(s, 2H), 1.70 (m, 2H), 1.40 (m, 2H), 1.35 (m, 2H), 1.24 (m, 6H), 0.83 (t,
J = 7.2 Hz, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 160.7, 159.8, 154.6,
150.1, 148.6, 145.4, 137.6, 109.0, 107.4, 106.8, 103.1, 56.4, 55.8, 40.8,
31.2, 28.8, 28.7, 26.6, 22.1, 13.9; ESI-MS (m/z) calcd for C23H32N5O2
[M + H]+ 410.26, found 410.33.
2H), 3.91 (s, 6H), 3.66 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H); 13
C
NMR (150 MHz, DMSO‑d6) δ 161.2, 159.5, 154.5, 150.4, 148.3, 145.6,
135.7, 110.2, 107.0, 106.7, 103.0, 56.2, 55.8, 35.7, 14.2; ESI-MS (m/z)
calcd for C17H20N5O2 [M + H]+ 326.16, found 326.22.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 53.75; H, 5.84; N, 18.44; Empirical
formula C17H19N5O2∙HCl⋅H2O: C, 53.75; H, 5.90; N, 18.22.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 57.50; H, 7.24; N, 14.51; Empirical
formula C23H31N5O2∙HCl⋅2H2O: C, 57.31; H, 7.53; N, 14.53.
3.32. 6,7-Dimethoxy-N4-isopropyl-N2-(pyridin-4-yl)quinazoline-2,4-
diamine (25):
3.37. 6,7-Dimethoxy-N4-(1-phenylethyl)–N2-(pyridin-4-yl)quinazoline-
2,4-diamine (30):
The title compound was prepared according to general procedures A
and B, and purified according to method C (60% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.28 (t, J = 7.4 Hz, 2H), 8.93 (s, 2H), 8.50 (d, J =
7.8 Hz, 1H), 7.88 (s, 1H), 7.04 (d, J = 7.2 Hz, 2H), 4.66 (m, 1H), 3.94 (s,
6H), 1.36 (t, J = 7.2 Hz, 6H); 13C NMR (150 MHz, DMSO‑d6) δ 160.7,
158.9, 154.7, 150.2, 148.7, 145.6, 137.8, 109.1, 107.3, 106.9, 103,1,
56.5, 55.8, 42.8, 22.1; ESI-MS (m/z) calcd for C18H22N5O2 [M + H]+
340.18, found 340.24.
The title compound was prepared according to general procedures A
and B, and purified according to method C (56% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.19 (d, J = 7.8 Hz, 2H), 9.13 (d, J = 7.8 Hz, 1H),
8.95 (s, 2H), 8.06 (s, 1H), 7.59 (d, J = 7.2 Hz, 2H), 7.35 (t, J = 7.8 Hz,
2H), 7.23 (t, J = 7.2 Hz, 1H), 7.01 (d, J = 4.8 Hz, 2H), 5.68 (m, 1H), 3.98
(s, 3H), 3.93 (s, 3H), 1.69 (d, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
DMSO‑d6) δ 160.6, 159.0, 154.8, 149.8, 148.8, 145.8, 144.7, 137.6,
128.4, 126.8, 126.3, 109.0, 107.3, 106.9, 103.2, 56.6, 55.9, 50.3, 22.3;
ESI-MS (m/z) calcd for C23H24N5O2 [M + H]+ 402.19, found 402.27.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 53.58; H, 5.88; N, 13.80; Empirical
formula C23H23N5O2∙2HCl⋅2H2O: C, 54.01; H, 5.91; N, 13.69.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 47.32; H, 5.95; N, 15.34; Empirical
formula C18H21N5O2∙2HCl∙5/2H2O: C, 47.27; H, 6.17; N, 15.32.
3.33. N4-n-Butyl-6,7-dimethoxy-N2-(pyridin-4-yl)quinazoline-2,4-
diamine (26):
The title compound was prepared according to general procedures A
and B, and purified according to method C (58% overall yield); 1H NMR
(500 MHz, CD3OD) δ 9.10 (dd, J = 18.6, 7.2 Hz, 2H), 7.33 (d, J = 13.2
Hz, 1H), 6.91 (d, J = 18.3 Hz, 1H), 6.84–6.81 (m, 2H), 3.84 (t, J = 5.2
Hz, 6H), 3.55–3.50 (m, 2H), 3.25–3.21 (m, 2H), 1.68–1.62 (m, 2H),
1.42–1.38 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, CD3OD)
δ 138.9, 110.0, 56.9, 56.6, 49.5, 49.3, 49.2, 49.0, 48.8, 48.7, 48.5, 42.4,
32.2, 21.4, 14.3; ESI-HRMS (m/z) calcd for C19H24N5O2 [M + H]+
354.1930, found 354.1930.
3.38. 6,7-Dimethoxy-N4-(2-methylphenyl)–N2-(pyridin-4-yl)
quinazoline-2,4-diamine (31):
The title compound was prepared according to general procedures A
and B, and purified according to method C (54% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 10.12 (s, 1H), 8.91 (d, J = 7.2 Hz, 2H), 8.76 (s,
2H), 8.02 (s, 1H), 7.44 (dd, J = 18.0, 7.2 Hz, 2H), 7.33 (m, 2H), 6.92 (s,
2H), 3.97 (s, 6H), 2.26 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 160.5,
159.1, 155.2, 150.0, 149.2, 146.3, 137.3, 136.2, 134.5, 130.6, 127.3,
126.8, 126.3, 109.1, 107.4, 106.9, 103.0, 56.3, 56.0, 17.9; ESI-MS (m/z)
calcd for C22H22N5O2 [M + H]+ 388.18, found 388.25.
3.34. 6,7-Dimethoxy-N4-(1-methylpropyl)–N2-(pyridin-4-yl)
quinazoline-2,4-diamine (27):
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 62.09; H, 5.38; N, 16.46; Empirical
formula C22H21N5O2∙HCl: C, 62.34; H, 5.23; N, 16.52.
The title compound was prepared according to general procedures A
and B, and purified according to method C (60% overall yield); 1H NMR
(500 MHz, CD3OD) δ 9.29 (d, J = 7.4 Hz, 2H), 7.63 (s, 1H), 7.12 (s, 1H),
6.97 (d, J = 8.0 Hz, 2H), 4.50 (q, J = 6.7 Hz, 1H), 3.97 (s, 6H), 1.84–1.78
(m, 1H), 1.77–1.71 (m, 1H), 1.38 (t, J = 6.3 Hz, 3H), 1.03 (t, J = 7.4 Hz,
3H); 13C NMR (125 MHz, CD3OD) δ 162.8, 161.4, 156.7, 151.7, 151.0,
147.7, 139.2, 110.2, 109.2, 108.5, 103.4, 56.9, 50.3, 49.7–48.7, 30.4,
20.5, 11.5; ESI-HRMS (m/z) calcd for C19H24N5O2 [M + H]+ 354.1930,
found 354.1944.
3.39. 6,7-Dimethoxy-N4-(o-trifluoromethylphenyl)–N2-(pyridin-4-yl)
quinazoline-2,4-diamine (32):
The title compound was prepared according to general procedures A
and B, and purified according to method B (52% overall yield); 1H NMR
(500 MHz, CD3OD) δ 9.00 (d, J = 8.0 Hz, 2H), 7.90–7.62 (m, 6H), 7.30
(s, 1H), 6.86 (d, J = 8.0 Hz, 2H), 4.02–4.00 (m, 6H); 13C NMR (125 MHz,
CD3OD) δ 162.7, 162.0, 157.5, 151.6, 151.2, 148.7, 138.8, 137.6, 134.3,
132.6, 129.1, 129.0, 128.1, 110.0, 109.0, 108.3, 102.8, 56.9, 49.4–48.5;
ESI-HRMS (m/z) calcd for C21H21N6O2 [M + H]+ 389.1726, found
389.1750.
3.35. 6,7-Dimethoxy-N4-(diethylmethyl)–N2-(pyridin-4-yl)quinazoline-
2,4-diamine (28):
The title compound was prepared according to general procedures A
and B, and purified according to method B (72% overall yield); 1H NMR
(500 MHz, CD3OD) δ 7.65 (s, 1H), 7.12 (s, 1H), 6.94–6.93 (d, J = 8.0 Hz,
2H), 4.50 (q, J = 6.7 Hz, 1H), 3.94 (s, 6H), 3.27–3.26 (m, 2H), 1.77–1.67
(m, 4H), 0.98–0.95 (m, 6H); 13C NMR (125 MHz, CD3OD) δ 162.7,
162.1, 156.6, 151.6, 150.9, 147.7, 139.1, 110.1, 109.0, 108.4, 103.1,
56.9, 56.6, 55.8, 28.4, 11.2; ESI-HRMS (m/z) calcd for C20H26N5O2 [M
+ H]+ 368.2087, found 368.2116.
3.40. 6,7-Dimethoxy-N4-(pyridin-3-ylmethyl)–N2-(pyridin-4-yl)
quinazoline-2,4-diamine (33):
The title compound was prepared according to general procedures A
and B, and purified according to method C (50% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.35 (s, 1H), 9.32 (d, J = 7.8 Hz, 2H), 8.79 (s,
2H), 8.74 (d, J = 2.4 Hz,1H), 8.48 (s, 1H), 7.87 (s, 1H), 7.83 (s, 1H), 7.38
10

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Bioorganic & Medicinal Chemistry 33 (2021) 116018
(dd, J = 8.4, 4.8 Hz, 1H), 6.99 (d, J = 7.8 Hz, 2H), 4.94 (d, J = 5.4 Hz,
2H), 3.94 (s, 3H), 3.92 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 160.7,
159.8, 155.0, 150.0, 149.2, 148.9, 148.4, 145.8, 137.9, 135.5, 134.7,
123.7, 109.1, 107.3, 106.9, 102.7, 56.2, 56.0, 41.8; ESI-MS (m/z) calcd
for C21H21N6O2 [M + H]+ 389.17, found 389.24.
3.45. 6,7-Dimethoxy-N4-diethylmethyl-N2-(pyridin-3-yl)quinazoline-
2,4-diamine (38):
The title compound was prepared according to general procedures A
and B, and purified according to method C (61% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.34 (s, 1H), 9.19 (d, J = 7.7 Hz, 1H), 8.56 (d, J
= 8.4 Hz, 1H), 8.04 (s, 1H), 7.89–7.85 (m, 2H), 6.96 (s, 2H), 4.45 (m,
1H), 3.98 (s, 3H), 3.96 (s, 3H), 1.74–1.72 (m, 4H), 0.96 (q, J = 7.2 Hz,
6H); 13C NMR (150 MHz, DMSO‑d6) δ 160.3, 154.9, 150.7, 149.6, 148.6,
145.3, 130.3, 127.5, 126.7, 123.8, 108.2, 107.3, 103.2, 56.7, 56.0, 54.1,
26.8, 10.7; ESI-MS (m/z) calcd for C20H26N5O2 [M + H]+ 368.21, found
368.28.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 48.92; H, 5.22; N, 16.23; Empirical
formula C21H20N5O2∙2HCl⋅3H2O: C, 48.94; H, 5.47; N, 16.30.
3.41. 6,7-Dimethoxy-N2-(pyridin-4-yl)-N4-(pyridin-4-ylmethyl)
quinazoline-2,4-diamine (34):
The title compound was prepared according to general procedures A
and B, and purified according to method C (54% overall yield); 1H NMR
(600 MHz, DMSO‑d6) 9.65 (t, J = 5.4 Hz, 1H), 9.19 (d, J = 7.8 Hz, 2H),
8.93 (s, 2H), 8.52 (d, J = 5.4 Hz, 2H), 7,98 (s, 1H), 7.49 (d, J = 6.0, 2H),
6.99 (d, J = 7.8 Hz, 2H), 4.91 (d, J = 5.4 Hz, 2H), 3.94 (s, 6H); 13C NMR
(150 MHz, DMSO‑d6) δ 160.7, 159.9, 155.0, 149.9, 149.6, 148.9, 148.3,
147.7, 137.7, 122.6, 109.1, 107.4, 106.9, 103.0, 56.3, 55.9, 43.1; ESI-
MS (m/z) calcd for C21H21N6O2 [M + H]+ 389.17, found 389.24.
The hydrochloride salt was prepared according to general procedure
C. Elemental analysis, found: C, 48.37; H, 5.29; N, 16.11; Empirical
formula C21H20N5O2∙2HCl⋅3H2O: C, 48.94; H, 5.47; N, 16.30.
3.46. 6,7-Dimethoxy-N4-(2-methylphenyl)–N2-(pyridin-3-yl)
quinazoline-2,4-diamine (39):
The title compound was prepared according to general procedures A
and B, and purified according to method C (53% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 10.31 (s, 1H), 9.10 (t, J = 2.4 Hz, 1H), 8.68 (d, J
= 6.0 Hz, 1H), 8.11 (s, 1H), 7.86–7.75 (m, 2H), 7.46–7.31 (m, 4H), 6.87
(s, 2H), 4.00 (s, 6H), 2.27 (s, 3H); 13C NMR (150 MHz, DMSO‑d6) δ
159.3, 155.4, 150.7, 150.0, 148.4, 146.0, 136.0, 134.5, 130.7, 130.3,
127.4, 127.3, 126.9, 126.4, 123.7, 108.4, 107.2, 103.3, 56.6, 56.1, 18.0;
ESI-MS (m/z) calcd for C22H22N5O2 [M + H]+ 388.18, found 388.25.
3.47. N4-Benzyl-6,7-dimethoxy-N2-(4-carbamoylpiperidyl)quinazolin-4-
3.42. 6,7-Dimethoxy-N4-ethyl-N2-(pyridin-3-yl)quinazoline-2,4-diamine
amine (40):
(35):
The title compound was prepared according to general procedures A
and B, and purified according to method C (63% overall yield). HPLC
purity was 99.8%; 1H NMR (500 MHz, CD3OD) δ 7.38–7.36 (t, J = 5.7
Hz, 3H), 7.29–7.26 (t, J = 7.5 Hz, 2H), 7.21–7.19 (d, J = 7.4 Hz, 1H),
6.89 (s, 1H), 4.00 (s, 11H), 6.80 (s, 1H), 4.75–4.70 (m, 4H), 3.90 (s, 3H),
3.87 (s, 3H), 2.91–2.87 (m, 2H), 3.86 (s, 3H), 2.48 (br, 1H), 1.80–1.78
(d, J = 10.4 Hz, 2H), 1.61–1.57 (m, 2H); 13C NMR (125 MHz, CD3OD)
δ180.7, 160.7, 158.8, 156.02, 147.3, 141.1, 129.4, 128.4, 127.9, 104.7,
104.6, 103.8, 56.7, 56.3, 49.5, 49.3, 49.2, 48.8, 48.65, 48.5, 45.7, 45.2,
44.1, 29.7; ESI-HRMS (m/z) calcd for C23H28N5O3 [M + H]+ 422.2192,
found 422.2186.
The title compound was prepared according to general procedures A
and B, and purified according to method C (59% overall yield); 1H NMR
(500 MHz, DMSO‑d6) δ 9.36 (s, 1H), 9.20 (d, J = 5.7 Hz, 1H), 9.01 (t, J =
4.9 Hz, 1H), 7.88 (dd, J = 7.7, 5.4 Hz, 2H), 7.83 (d, J = 8.0 Hz, 1H), 6.92
(s, 2H), 3.95 (s, 6H), 3.73 (q, J = 6.5 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H);
13C NMR (125 MHz, DMSO‑d6) δ 159.9, 155.1, 150.9, 149.8, 148.7,
145.3, 130.4, 127.7, 126.9, 124.1, 108.6, 107.4, 103.2, 56.7, 56.2,
40.2–39.2, 36.1, 14.5; ESI-HRMS (m/z) calcd for C17H20N5O2 [M + H]+
326.1617, found 326.1645.
3.43. 6,7-Dimethoxy-N4-isopropyl-N2-(pyridin-3-yl)quinazoline-2,4-
4. Biology
diamine (36):
4.1. Activity against P. Falciparum
The title compound was prepared according to general procedures A
and B, and purified according to method C (59% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.34 (d, J = 2.4 Hz, 1H), 9.21 (d, J = 7.0 Hz, 1H),
8.55 (d, J = 7.8 Hz, 1H), 7.88 (m, 2H), 7.78 (t, J = 7.2 Hz, 1H), 6.89 (s,
2H), 4.70 (m, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 1.37 (d, J = 7.7 Hz, 6H);
13C NMR (150 MHz, DMSO‑d6) δ 159.1, 154.9, 150.8, 149.6, 148.5,
145.3, 130.3, 127.5, 126.8, 123.9, 108.2, 107.3, 102.9, 56.5, 56.0, 43.0,
22.1; ESI-MS (m/z) calcd for C18H22N5O2 [M + H]+ 340.18, found
340.25.
In vitro activity against the erythrocytic stages of P. falciparum was
determined using a 3H-hypoxanthine incorporation assay22 with the
drug-sensitive NF54 strain.23 Compounds were dissolved in DMSO at 10
mg/mL and further diluted in medium before addition to parasite cul-
tures incubated in Roswell Park Memorial Institute (RPMI) 1640 me-
dium without hypoxanthine, supplemented with 4-(2-hydroxyethy)-1-
piperazineethanesulfonic acid (HEPES, 5.94 g/L), NaHCO3 (2.1 g/L),
neomycin (100 U/mL), AlbumaxR (5 g/L), and washed human red cells
A + at 2.5% hematocrit (0.3% parasitemia). Serial drug dilutions of
eleven 3-fold dilution steps covering a range from 100 to 0.002 μg/mL
3.44. N4-n-Butyl-6,7-dimethoxy-N2-(pyridin-3-yl)quinazoline-2,4-
were prepared. The 96-well plates were incubated in a humidified at-
diamine (37):
mosphere (4% CO2, 3% O2, 93% N2) at 37 ◦C. After 48 h, 50
hypoxanthine (=0.5
μ
L of 3H-
μ
Ci) was added to each well of the plate. The plates
The title compound was prepared according to general procedures A
and B, and purified according to method C (57% overall yield); 1H NMR
(600 MHz, DMSO‑d6) δ 9.35 (s, 1H), 9.18 (d, J = 7.0 Hz, 1H), 9.09 (t, J =
5.4 Hz, 2H), 7.97 (s, 1H), 7.88 (m, 2H), 6.97 (s, 2H), 3.98 (s, 3H), 3.95 (s,
3H), 3.68 (q, J = 6.6 Hz, 2H), 1.72 (m, 2H), 1,45 (m, 2H), 0.96 (t, J =
7.2 Hz, 3H); 13C NMR (150 MHz, DMSO‑d6) δ 159.9, 154.9, 150.7,
149.4, 148.5, 145.1, 130.2, 127.5, 126.6, 123.9, 108.4, 107.2, 103.2,
56.5, 56.0, 40.7, 30.7, 19.8, 13.9; ESI-MS (m/z) calcd for C19H24N5O2
[M + H]+ 354.19, found 354.27.
were incubated for a further 24 h under the same conditions. The cells
were then harvested from the plate using a Betaplate™ cell harvester
(Wallac, Zurich, Switzerland), and the red blood cells were transferred
onto a glass fiber filter and lysed with distilled water. The dried filters
were inserted into plastic foil with 10 mL of scintillation fluid and
counted in a Betaplate™ liquid scintillation counter (Wallac, Zurich,
Switzerland). IC50 values were calculated from sigmoidal inhibition
curves by linear regression and 4-parameter logistic regression using
GraphPad Prism S. Chloroquine (Sigma C6628) was used as a control.
11

Y. Mizukawa et al.
Bioorganic & Medicinal Chemistry 33 (2021) 116018
Assays were performed in at least two independent replicates.
Acknowledgments
We wish to thank Monica Cal, Romina Rocchetti, and Sonja Keller
(all Swiss TPH) for assistance with in vitro drug testing, and Dr. Saeko
Murakami and Dr. Nobuyoshi Aoki (both Kanagawa Institute of Indus-
trial Science and Technology, KISTEC) for analytical support. We also
thank Prof. Keisuke Suzuki (Tokyo Institute of Technology), Prof.
Masahiro Hirama (Tohoku University), and Dr. Sofia Elouali (GlyTech,
Inc. Kyoto, Japan) for manuscript suggestions.
4.2. In vitro cytotoxicity with L6 cells
Assays were performed in 96-well microtiter plates, each well con-
taining 100 µL of RPMI 1640 medium supplemented with 1% L-gluta-
mine (200 mmol) and 10% fetal bovine serum with 4000 L6 cells (a
primary cell line derived from rat skeletal myoblasts).24,25 Serial drug
dilutions of eleven 3-fold dilution steps covering a range from 100 to
0.002 μg/mL were prepared 24 h post L6 cell seeding. The plates were
Appendix A. Supplementary data
incubated for 70 h and inspected under an inverted microscope to
confirm sterile conditions and growth of the controls. Next, 10 µL of
resazurin solution (12.5 mg resazurin dissolved in 100 mL water) was
added to each well, and the plates were incubated for another 2 h. The
plates were then read using a Spectramax Gemini XS microplate fluo-
rometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) at an
excitation wavelength of 536 nm and emission wavelength of 588 nm.
The IC50 values were calculated by linear regression and 4-parameter
logistic regression from the sigmoidal dose-inhibition curves using
SoftmaxPro software (Molecular Devices Corp., Sunnyvale, CA, USA).
Supplementary data (NMR spectra are available free of charge.
Almost all compounds in Table 2 are available on demand.) to this
article can be found online at https://doi.org/10.1016/j.bmc.2021.11
6018.
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13